This proposal is designed to study the interactions between the apoenzyme of pig heart aspartate aminotransferase (AAT) and inhibitors of both reversible and irreversible types which interfere with the binding of the natural cofactors, pyridoxal 5'-phosphate (PLP) and pyridoxamine 5'-phosphate (PMP). The specific aims are as follows. First, the dissociation constant of apoAAT-PMP complex will be determined. Second, the affinity of the various reversible inhibitors for apoAAT will be evaluated. Third, the cofactor saturation of AAT in pig hearts will be measured. Fourth, the 2',3'-dialdehyde (dial) analogs of nucleotides will be surveyed to deduce the structural features required for apoAAT inactivation. Fifth, the time course of apoAAT inactivation by dial nucleotides will be established. Sixth, the fate of the functional groups on the dial nucleotides will be determined. Seventh, the amino acid residue modified by the reactive nucleotides will be identified. To achieve these goals, the following methodology will be adopted. The dissociation constants for PMP and the reversible inhibitors will be evaluated by several versions of kinetic assay and centrifugal ultrafiltration. Cofactor saturation of AAT in vivo will be determined by assaying the enzyme in the presence and absence of added PLP. The many commercial dial nucleotides and their derivatives will be examined for their potential to inactivate apoAAT. Dial nucleotide inactivation of apoAAT will be followed by first incubating apoAAT with dialAMP and then stopping the reaction and activating the unreacted enzyme by adding PLP at various time intervals. Loss of 5'-phosphate and 1'-nitrogen base from dialAMP will be ascertained and quantitated by inorganic phosphate determination and chromatographic isolation respectively. Radioactive compounds will be used for confirmation. Finally, the site of inactivation will be identified by first comparing the high pressure liquid chromatographic elution profile of proteolytic enzyme digests of the native and inactivated apoAAT. The unique peptide will then be analyzed for its amino acid composition. The identity and position of the residue modified will be deduced from the known sequence of AAT. AAT is found in abundance in the heart where it is believed to be intimately involved in the production of energy. In vitro at least, AAT seems free from sophisticated mechanisms of regulation. This study represents a probe into the possibility of regulation at the level of apoenzyme-cofactor combination.